The present invention relates to a laser scan microscope for forming an image of a sample on the basis of density information obtained by two-dimensionally scanning a sample by means of a finely-focused or point-illuminating laser beam. The present invention also relates to a light-measurement apparatus which can be incorporated in the photoelectric conversion section of the laser scan microscope.
A laser scan microscope irradiates a sample with a point-illuminating laser beam while simultaneously scanning the laser beam over the sample in X-axis and Y-axis directions by means of an object lens. The transmitted light, reflected light and fluorescent light coming from the sample are made to pass through the object lens and an optical system of the microscope, and are then detected by a detector, thereby obtaining the density information on the two-dimensional image.
The laser scan microscope forms an image representing the two-dimensional distribution of the density information with reference to the X-Y scan positions, and displays that image on the screen of a CRT display as a luminescent spot distribution. A confocal laser scan microscope is one type of the laser scan microscope. The confocal laser scan microscope comprises a diaphragm arranged at a position which is conjugate with respect to the sample of a detection optical system and having an aperture which is smaller than the diffraction limit of illumination light or measurement light.
A typical structure of a detection system employed in a laser scan microscope will be described. The detection system comprises a detector for photoelectrically converting light coming from a sample, such as transmitted light, reflected light and fluorescent light, into an electric signal. The detector is made of a photodiode, a photomultiplier, or the like and is designed to have appropriate sensitivity. An output voltage (current) signal which is produced from the detector in accordance with the amount or intensity of light is supplied to an amplifier, for amplification.
An output signal of the amplifier includes two kinds of components: an offset component which takes a constant value without reference to the position of an image (such as background light and an electrical offset); and a signal component which varies in accordance with a light intensity distribution. The former component is removed from the output signal by an offset subtraction circuit since it is often the cause of a narrow dynamic range in measurement. The latter component is converted into digital data by an A/D converter circuit, and the resultant digital data is stored in a memory and used as a measurement value.
Image data obtained in this manner represents a light intensity and a light intensity distribution. To be more specific, the density of the image indicates the intensity of an output signal, i.e., the intensity of the detected light, while the two-dimensional components of the image indicates the distribution of the light intensity.
In this type of laser scan microscope, a variety of measurement parameters, such as the sensitivity of the detector, the amplification factor of the amplifier, and the offset amount of the offset subtraction circuit, have to be properly adjusted to produce an image with optimally sharp contrast. If this adjustment is not properly made, various problems are brought about. For example, if the offset amount is not appropriately adjusted, the offset component may correspond to most of the dynamic range of the A/D converter circuit. Conversely, even the signal component may be removed from the image signal.
If the sensitivity of the photoelectric converter and the amplification factor of the amplifier are too high or large, the signal may exceed the upper limit of the dynamic range of the A/D converter circuit, resulting in saturation; conversely, if they are too low or small, the amplitude of the signal may be so low that an image showing a subject under measurement may not be formed.
In the conventional laser scan microscope, therefore, the measurement parameters have to be adjusted one by one while simultaneously looking at the image that is being formed at the time.
However, this adjustment method is disadvantageous in that a long time is required for completing the adjustment of all parameters. In addition, the adjustment requires a certain degree of skill. For example, when the fluorescent light coming from a sample is measured, the measurement parameters have to be adjusted in a short time. If this adjustment is not made in a short time, the sample under measurement may be damaged in the meantime, in addition, the throughput of the measurement may be lowered.
Jpn. Pat. Appln. KOKAI Publication No. 8-160304 discloses a technique for automatically optimizing measurement parameters. According to this publication, a sample is measured in the state where the sensitivity of a photoelectric converter, the amplification factor of an amplifier and the offset amount of an offset subtraction circuit are set to be appropriate, and the resultant measurement data is used to obtain optimal parameters. Although this method offers a comparatively short measurement time and variations in measurement data can be suppressed, a certain time is inevitably required before the start of actual measurement. In addition, the sample may be damaged before it is actually measured.
Jpn. Pat. Appln. KOKAI Publication No. 6-303506 discloses a technique for producing a contrast emphasis effect, a correction effect or the like by varying measurement parameters in synchronism with the sampling clocks, i.e., by dynamically varying the measurement parameters for each of the pixels of the image. However, even the technique of the reference does not eliminate the need to predetermine parameter values and desirable positions. It is not possible for the technique of the reference to determine optimal values unless an image is formed and measured.